Effect Of Chronic Ethanol Ingestion Research Articles

Chronic Ethanol Intake Impairs Niacin Nutritional Status in Mice.

Niacin is involved in many biological reactions relating energy metabolism, redox reactions, DNA repair and longevity. Since niacin deficiency has been reported in alcoholic patients, and niacin coenzyme NAD is used as substrate to dehydrogenate ethanol in the liver, ethanol consumption can be a factor to impair niacin nutritional status. We have recently established the niacin insufficient model mice using kynurenine 3-monooxygenase knock out (KMO-/-) mice with niacin-limited diet, which lack the de novo NAD synthesis pathway from tryptophan. To evaluate the effects of chronic ethanol intake on niacin nutritional status, 4 wk old KMO-/- mice were fed 4 or 30 mg/kg nicotinic acid containing diets with or without 15% ethanol for 35 d. The mice fed 4 mg/kg nicotinic acid diet with ethanol showed lower body weight gain and niacin nutritional markers such as liver and blood NAD, and urine nicotinamide metabolites than the mice without ethanol. These animals did not show any difference in the NAD synthesis, NAD salvage and nicotinamide catabolic pathways. Chronic ethanol intake failed to affect any indices in the mice fed the 30 mg/kg nicotinic acid diet. When the diet was exchanged the 4 mg/kg for 30 mg/kg nicotinic acid diet to the mice showed chronic ethanol-induced growth retardation, their body weight rapidly increased. These results show that chronic ethanol intake impairs niacin nutritional status in the niacin insufficient mice, and enough niacin intake can prevent this impairment. Our findings also suggest that chronic ethanol intake increases niacin requirement by increase of NAD consumption.

Effect of chronic ethanol ingestion on dendritic cell population during oral mucosal repair: An experimental study.

The aim of this study was to analyze the effect of chronic ethanol ingestion on dendritic cell repopulation during the repair process of rat oral mucosa and in the rat spleen by analyzing the immunohistochemical expression of dendritic cell markers. Wistar rats ingested 20% ethanol solution for 28 days; a surgical wound was performed on the rat tongue after this period. The repair process and the number of CD1a+, CD11c+, and CD207+ cells in the regions adjacent to the wound were determined at day 1, 3, and 7 following the wound as well as in the rat spleen. The wound-only group (no ethanol exposure) had complete reepithelization after 7 days, but this did not occur in the ethanol + wound group at this time point. The inflammatory infiltrate was significantly reduced in animals exposed to ethanol, which also showed significantly lower counts of CD1a+, CD11c+, and CD207+ cells than the wound-only group at all experimental time points. In addition, ethanol exposure also resulted in lower densities of CD11c+ and CD207+ cells in the rat spleen. In conclusion, chronic ethanol intake had a negative impact on dendritic cell numbers, a fact that may contribute to delay in oral mucosa repair.

Effects of Chronic Ethanol Intake on High‐Fat Diet Induced Metabolic Dysfunction in Mice

High‐fat diet (HFD) induced obesity is associated with insulin resistance and is a major risk factor for numerous co‐morbidities such as type II diabetes. Conflicting clinical and preclinical evidence suggests that moderate alcohol consumption improves insulin sensitivity in obese subjects, and therefore may act to protect against obesity‐related metabolic comorbidities. However, the impact of free choice ethanol (EtOH) intake and HFD consumption on insulin action and glucose homeostasis in animal models remains unclear. Therefore, the overall goal of these studies was to determine if long‐term EtOH intake may alter insulin and glucose function in a mouse model of HFD induced obesity. Six‐week old male C57Bl/6J mice were given ad libitum access to HFD (n=10; 60% calories from fat) or control diet (Chow, n=10; 10% calories from fat) for two weeks. Mice were then given increasing volumes of EtOH (3%, 7%, and 10%) over one week as an EtOH‐ramp initiation period for subsequent 24‐hour access to a two‐bottle choice of water or 10% EtOH (vol/vol, in water). Body mass, EtOH and water intake, and EtOH preference was assessed every 24 hours for six weeks. HFD mice had significantly higher body mass compared to Chow mice (p<0.001). By the end of the study, Chow mice consumed significantly more EtOH than mice on HFD (p<0.001) and had higher preference towards EtOH (p<0.001). At the end of treatment, we measured 4‐hour fasting glucose levels and performed insulin tolerance tests (to examine the ability of intraperitoneal insulin to suppress blood glucose levels) in half of the mice in each group. These metabolic data were compared to weight and aged matched mice given HFD or Chow without EtOH access. Insulin sensitivity was significantly reduced in HFD mice (p=0.003; two‐way ANOVA), and EtOH consumption did not alter HFD‐induced insulin resistance. Furthermore, there appeared to be a trend for increased 4 hour fasting glucose levels in HFD (185.8±9.4), HFD+EtOH (176±25.9) and Chow+EtOH (177.8±21.6) groups compared to Chow alone (156.4±6.2). Together, these results suggest that moderate EtOH drinking in a two‐bottle choice model does not improve insulin sensitivity in HFD mice, but that higher levels of EtOH intake in control diet mice may cause impaired glucose homeostasis that may not be due to changes in insulin sensitivity.Support or Funding InformationPenn State Dept of Neural and Behavioral Science, NIH grants AA022937 and HL122507This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A novel comparative pattern analysis approach identifies chronic alcohol mediated dysregulation of transcriptomic dynamics during liver regeneration.

BackgroundLiver regeneration is inhibited by chronic ethanol consumption and this impaired repair response may contribute to the risk for alcoholic liver disease. We developed and applied a novel data analysis approach to assess the effect of chronic ethanol intake in the mechanisms responsible for liver regeneration. We performed a time series transcriptomic profiling study of the regeneration response after 2/3rd partial hepatectomy (PHx) in ethanol-fed and isocaloric control rats.ResultsWe developed a novel data analysis approach focusing on comparative pattern counts (COMPACT) to exhaustively identify the dominant and subtle differential expression patterns. Approximately 6500 genes were differentially regulated in Ethanol or Control groups within 24 h after PHx. Adaptation to chronic ethanol intake significantly altered the immediate early gene expression patterns and nearly completely abrogated the cell cycle induction in hepatocytes post PHx. The patterns highlighted by COMPACT analysis contained several non-parenchymal cell specific markers indicating their aberrant transcriptional response as a novel mechanism through which chronic ethanol intake deregulates the integrated liver tissue response.ConclusionsOur novel comparative pattern analysis revealed new insights into ethanol-mediated molecular changes in non-parenchymal liver cells as a possible contribution to the defective liver regeneration phenotype. The results revealed for the first time an ethanol-induced shift of hepatic stellate cells from a pro-regenerative phenotype to that of an anti-regenerative state after PHx. Our results can form the basis for novel interventions targeting the non-parenchymal cells in normalizing the dysfunctional repair response process in alcoholic liver disease. Our approach is illustrated online at http://compact.jefferson.edu.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2492-x) contains supplementary material, which is available to authorized users.

Activation of NF‐κB after chronic ethanol intake and haemorrhagic shock/resuscitation in mice

Chronic ethanol abuse and haemorrhagic shock are major causes of global mortality and, separately, induce profound hepato- and immune-toxic effects via activation of NF-κB. Here, we assessed the effects of chronic ethanol intake upon the pathophysiological derangements after haemorrhagic shock with subsequent resuscitation (H/R), with particular attention to the contribution of NF-κB. Transgenic NF-κB(EGFP) mice, expressing the enhanced green fluorescent protein (EGFP) under the transcriptional control of NF-κB cis-elements were fed a Lieber-DeCarli diet containing ethanol (EtOH-diet) or an isocaloric control diet for 4 weeks and were then pairwise subjected to H/R. Liver tissues and peripheral blood were sampled at 2 or 24 h after H/R. Cytokines in blood and tissue and leukocyte activation (as CD11b expression) were measured, along with EGFP as a marker of NF-κB activation. The EtOH-diet increased mortality at 24 h after H/R and elevated liver injury, associated with an up-regulation of NF-κB-dependent genes and IL-6 release; it also increased production of NF-κB-driven intercellular adhesion molecule 1 (ICAM-1) and EGFP in liver tissue. At 2h after the H/R procedure in ethanol-fed mice we observed the highest proportion of NF-κB activated non-parenchymal cells and an NF-κB-dependent increase in polymorphonuclear leukocyte CD11b expression. The EtOH-diet exacerbated liver injury after H/R, accompanying an overwhelming hepatic and systemic immune response. Our findings contribute to evidence implicating NF-κB as a key player in the orchestration of the immune response in haemorrhagic shock patients with a history of chronic ethanol abuse.

Effect of Bacterial Pneumonia on Lung Simian Immunodeficiency Virus (SIV) Replication in Alcohol Consuming SIV‐Infected Rhesus Macaques

Opportunistic infections in human immunodeficiency virus (HIV)-infected persons have been shown to increase the rate of HIV replication. In populations where prophylaxis against Pneumocystis pneumonia is utilized, bacterial pneumonia is now the leading cause of lower respiratory tract infection in HIV+ patients. Our prior studies have shown that chronic alcohol consumption in demarcated simian immunodeficiency virus (SIV)-infected rhesus macaques increases plasma viral load set point and accelerates progression to end-stage acquired immune deficiency syndrome. While chronic alcohol abuse is well known to increase the incidence and severity of bacterial pneumonia, the impact of alcohol consumption on local and systemic SIV/HIV burden during lung infection is unknown. Therefore, we utilized the macaque SIV infection model to examine the effect of chronic ethanol (EtOH) feeding on SIV burden during the course of pulmonary infection with Streptococcus pneumoniae, the most commonly identified etiology of bacterial pneumonia in HIV+ and HIV- persons in developed countries. Alcohol was administered starting 3months before SIVmac251 inoculation to the end of the study via an indwelling intragastric catheter to achieve a plasma alcohol concentration of 50 to 60mM. Control animals received isocaloric sucrose. Four months after SIV infection, the right lung was inoculated with 2×10(6) CFU S.pneumoniae. Leukocyte recruitment into the lung, pulmonary bacterial clearance, and clinical course were similar between EtOH and control groups. While plasma SIV viral load was similar between groups postpneumonia, chronic EtOH-fed macaques showed a prolonged increase in SIV RNA in bronchoalveolar lavage fluid. Alveolar macrophages isolated from EtOH-fed macaques 1day post-pneumonia showed greater nuclear factor kappa beta (NF-κB) activation. This study indicates that chronic EtOH feeding results in enhanced local, but not systemic, SIV replication following pneumococcal pneumonia. Increased NF-κB activity in the setting of chronic EtOH ingestion may play a mechanistic role in this observation.

Influence of chronic ethanol intake on mouse synaptosomal aspartyl aminopeptidase and aminopeptidase A: Relationship with oxidative stress indicators

Aminopeptidase A (APA) and aspartyl aminopeptidase (ASAP) not only act as neuromodulators in the regional brain renin-angiotensin system, but also release N-terminal acidic amino acids (glutamate and aspartate). The hyperexcitability of amino acid neurotransmitters is responsible for several neurodegenerative processes affecting the central nervous system. The purpose of the present work was to study the influence of chronic ethanol intake, a well known neurotoxic compound, on APA and ASAP activity under resting and K+-stimulated conditions at the synapse level. APA and ASAP activity were determined against glutamate- and aspartate-β-naphthylamide respectively in mouse frontal cortex synaptosomes and in their incubation supernatant in a Ca2+-containing or Ca2+-free artificial cerebrospinal fluid. The neurotoxic effects were analyzed by determining free radical generation, peroxidation of membrane lipids and the oxidation of synaptosomal proteins. In addition, the bioenergetic behavior of synaptosomes was analyzed under different experimental protocols. We obtained several modifications in oxidative stress parameters and a preferential inhibitor effect of chronic ethanol intake on APA and ASAP activities. Although previous in vitro studies failed to show signs of neurodegeneration, these in vivo modifications in oxidative stress parameters do not seem to be related to changes in APA and ASAP, invalidating the idea that an excess of free acidic amino acids released by APA and ASAP induces neurodegeneration.

Animal models for studying hepatitis C and alcohol effects on liver

Chronic consumption of ethanol has a dramatic effect on the clinical outcome of patients with hepatitis C virus (HCV) infection, but the mechanism linking these two pathologies is unknown. Presently, in vitro systems are limited in their ability to study the interaction between a productive wild-type HCV infection and chronic ethanol exposure. Mouse models are potentially very useful in dissecting elements of the HCV-ethanol relationship. Experiments in mice that transgenically express HCV proteins are outlined, as are experiments for the generation of mice with chimeric human livers. The latter models appear to have the most promise for accurately modeling the effects of chronic ethanol intake in HCV-infected human livers.

Reduced levels of folate transporters (PCFT and RFC) in membrane lipid rafts result in colonic folate malabsorption in chronic alcoholism

We studied the effect of chronic ethanol ingestion on folate transport across the colonic apical membranes (CAM) in rats. Male Wistar rats were fed 1 g/kg body weight/day ethanol (20%) solution orally for 3 months and folate transport was studied in the isolated colon apical membrane vesicles. The folate transport was found to be carrier mediated, saturable, with pH optima at 5.0. Chronic ethanol ingestion reduced the folate transport across the CAM by decreasing the affinity of transporters (high Km) for the substrate and by decreasing the number of transporter molecules (low Vmax) on the colon luminal surface. The decreased transport activity at the CAM was associated with down-regulation of the proton-coupled folate transporter (PCFT) and the reduced folate carrier (RFC) which resulted in decreased PCFT and RFC protein levels in the colon of rats fed alcohol chronically. Moreover, the PCFT and the RFC were found to be distributed in detergent insoluble fraction of the CAM in rats. Floatation experiments on Optiprep density gradients demonstrated the association of the PCFT and the RFC protein with lipid rafts (LR). Chronic alcoholism decreased the PCFT and the RFC protein levels in the CAM LR in accordance with the decreased synthesis. Hence, we propose that downregulation in the expression of the PCFT and the RFC in colon results in reduced levels of these transporters in colon apical membrane LR as a mechanism of folate malabsorption during chronic alcoholism.

Chronic ethanol consumption up-regulates protein-tyrosine phosphatase-1B (PTP1B) expression in rat skeletal muscle

to investigate the potential effects of chronic ethanol intake on protein-tyrosine phosphatase-1B (PTP1B) and the insulin receptor signaling pathway in rat skeletal muscle. rats received ethanol treatment at a daily dose of 0 (control), 0.5 (group L), 2.5 (group M) or 5 gxkg(-1) (group H) via gastric gavage for 22 weeks. In vivo insulin sensitivity was measured using a hyperinsulinemic-euglycemic clamp. Expression of PTP1B in skeletal muscles was examined at both the mRNA (real-time PCR) and protein (Western blot) levels. PTP1B activity was assayed with a p-nitrophenol phosphate (PNPP) hydrolysis method. Changes of insulin signaling in skeletal muscle were analyzed with Western blotting. the activity and expression of PTP1B were dose-dependently elevated 1.6 and 2.0 fold in the skeletal muscle by ethanol, resepctively, at the doses of 2.5 and 5 gxkg(-1)xd(-1). Total IRβ and IRS-1, as well as their phosphorylated forms, were decreased by ethanol at the two higher doses. Moreover, chronic ethanol consumption resulted in a significant inhibition of the association between IRS-1 and the p85 subunit of phosphatidylinositol 3-kinase, inhibition of Akt phosphorylation and reduced levels of mitogen-activated protein kinase phosphorylation. chronic ethanol intake at 2.5 and 5 xkg(-1)xd(-1) sufficient doses can down-regulate the expression of IRβ, P-IRβ, and IRS-1, as well as the phosphorylated forms of IRS-1 and Akt, in rat skeletal muscle, possibly through increased PTP1B activity.

Modulatory effects of chronic ethanol ingestion on hepatotoxicity-related parameters in male rats

5 g of ethanol (20% w/v) per kg body weight was administered to ten rats weighing between 166 – 170 grams daily for 28 days along side with normal feeds and water ad libitum. The administration of ethanol was done orally using oral catheter. A control group o ten rats was set up for a proper experimental evaluation. Analysis at the end of the adminstration showed tha ethanol in a dose of 5g/kg body weight significantly modulates (P Keywords: Rats, Ethanol, Oral administration, Catheter, Liver, Hepatotoxicity, Antioxidant enzyme

O efeito do consumo crônico de etanol na absorção duodenal de ferro em camundongos

Alcoholists present an increase of iron hepatic concentration, although the responsible mechanisms for this deposition are still unknown. Despite the extensive literature related on the iron absorption in different pathological conditions, the effect of chronic ethanol consumption are still not conclusive and not completely understood. To verify the effect of chronic ethanol ingestion on duodenal absorption of iron. Ten male Swiss mice were divided into two groups: group 1 (n = 5) - control, and group 2 (n = 5) - water consumption with ethanol, as only water source. The animals were followed during 120 days. After this period, the duodenum was isolated and saline solution containing ascorbate of iron II in the 0,016 concentration of mg of iron element was infused. The effluent was collected in times 20, 40, 60, 80, 100 and 120 minutes. The results were analyzed by Mann-Whitney and Kruskal-Wallis tests. The significance was set for P<0.05. No difference was found between iron absorption as well as iron absorption curves in groups 1 and 2. The chronic consumption of ethanol did not alter iron absorption.

Effect of chronic ethanol exposure on the liver of Clock-mutant mice

In humans, chronic ethanol consumption leads to a characteristic set of changes to the metabolism of lipids in the liver that is referred to as an "alcoholic fatty liver (AFL)". In severe cases, these metabolic changes result in the enlargement and fibrillization of the liver and are considered risk factors for cirrhosis and liver cancer. Clock-mutant mice have been shown to display abnormal lipid metabolism and alcohol preferences. To further understand the potential interactions between ethanol consumption, lipid metabolism, and the circadian clock, we investigated the effect of chronic ethanol intake on the lipid metabolism of Clock-mutant mice. We found that ethanol treatment produced a number of changes in the liver of Clock-mutant mice without impacting the wild-type controls. First, we found that 8 weeks of exposure to ethanol in the drinking water increased the weight of the liver in Clock-mutant mice. Ethanol treatment also increased triglyceride content of liver in Clock-mutant and wild-type mice. This increase was larger in the mutant mice. Finally, ethanol treatment altered the expression of a number of genes related to lipid metabolism in the Clock-mutant mice. Interestingly, this treatment did not impact circadian clock gene expression in the liver of either genotype. Thus, ethanol produces a number of changes in the liver of Clock-mutant mice that are not seen in the wild-type mice. These changes are consistent with the possibility that disturbance of circadian rhythmicity associated with the Clock mutation could be a risk factor for the development of an alcoholic fatty liver.

Chronic ethanol consumption resulting in the downregulation of insulin receptor-β subunit, insulin receptor substrate-1, and glucose transporter 4 expression in rat cardiac muscles

The objective of this study was to investigate the effect of chronic ethanol intake on the expression of insulin receptor β subunit (IR-β), insulin receptor substrate-1 (IRS-1), and glucose transporter 4 (Glut4) in rat cardiac muscle. Forty-eight male Wistar rats were randomly classified into four groups and to each group, ethanol was administered daily at the respective doses of 0 (control, C), 0.5 g kg −1 (low ethanol, L), 2.5 g kg −1 (middle ethanol, M), and 5 g kg −1 (high ethanol, H). Twenty-two weeks later, the rats were anesthetized, and the left ventricle muscles were excised. The IR-β, IRS-1, and Glut4 mRNA levels in the cardiac muscle tissues were detected by reverse-transcription polymerase chain reaction (RT-PCR); The IR-β, tyrosine phosphorylation of IR-β (PY-IR-β), IRS-1, tyrosine phosphorylation of IRS-1 (PY-IRS-1), and Glut4 protein levels were measured by Western blotting. Compared to the control group, the IR-β, IRS-1, and Glut4 mRNA levels in groups H and M decreased remarkably. In addition, the protein levels of IR-β, IRS-1, and Glut4 showed a corresponding decline in ethanol-treated groups, especially in group H. Moreover, the PY-IR-β and PY-IRS-1 protein levels decreased in the hearts of ethanol-treated rats with corresponding changes in the IR-β and IRS-1 protein levels. The present study showed that chronic ethanol intake could downregulate the expression levels of IR-β, IRS-1, and Glut4 in rat cardiac muscles.

Structural evaluation of the effects of chronic ethanol ingestion on the testis of Calomys callosus

Chronic exposure to ethanol may results in pathophysiologic changes in cellular function. The present work was designed to investigate the morphology of testis submitted to experimental ethanol ingestion. Experimental animals were divided into two groups. The control group ( n = 23) received a solid diet and tap water and the alcoholic group ( n = 23) received the same solid diet and ethanol P.A. diluted 20% in water (v/v). After 120 days of treatment, all animals were anesthetized, weighed and sacrificed. Testosterone and luteinizing hormone levels in serum were lower in the alcoholic group than in the control group. Histological and ultrastructural alterations were observed in the testicular alcoholic germinative cells like enormous spaces, lipid droplets accumulation, digestive vacuoles, irregular diameter of the seminiferous tubules and interstitial dilated blood vessels. It was concluded that 20% ethanol provokes lesions on the testis germinative epithelium probably inducing gonadal dysfunction.

Chronic ethanol intake-induced changes in open-field behavior and cal-cium/calmodulin-dependent protein kinase IV expression in nucleus accumbens of rats: naloxone reversal

To investigate the effects of chronic ethanol intake on the locomotor activity and the levels of calcium/calmodulin-dependent protein kinase IV (CaM kinase IV) in the nucleus accumbens (NAc) of rats. Simultaneously, the effects of nonselective opioid antagonist (naloxone) on the CaM kinase IV expression in the NAc and ethanol consumption of rats were also observed. Ethanol was administered in drinking water at the concentrations of 6% (v/v), for 28 d. The locomotor activity of rats was investigated in the open-field apparatus. CaM kinase IV levels in the NAc were analyzed using Western blotting. Rats consuming ethanol solution exhibited a significant decrease of ambulation activity, accompanied by a reduced frequency of explorative rearing in an open-field task on d 7 and d 14 of chronic ethanol ingestion, whereas presumed adaptation to the neurological effects of ethanol was observed on d 28. Chronic ethanol intake elicited a significant decrease of the CaM kinase IV expression in the nuclei, but not in the cytoplasm of the NAc on d 28. Naloxone treatment significantly attenuated ethanol intake of rats and antagonized the decrease of CaM kinase IV in the nuclei of NAc neurons. The cytosolic CaM kinase IV protein levels of the NAc also increased in rats exposed to ethanol plus naloxone. Chronic ethanol intake-induced changes in explorative behavior is mediated at least partly by changes in CaM kinase IV signaling in the nuclei of the NAc, and naloxone attenuates ethanol consumption through antagonizing the downregulation of CaM kinase IV in the NAc.

Chronic ethanol ingestion alters xenobiotic absorption through the skin: Potential role of oxidative stress

Alcohol ingestion is correlated with several skin disorders and it has been proposed that changes in skin properties may be an early indicator of alcohol misuse. Topically applied ethanol is an effective transdermal penetration enhancer; however, little is known about the effects of chronic ethanol ingestion on skin. Rats were pair fed a diet containing 36% ethanol for twelve weeks. The animals were then switched to a non-ethanol diet and were monitored for up to four weeks. Non-invasive measurements for changes in dermal blood flow using laser Doppler velocimetry (LDV), damage to skin barrier via transepidermal water loss (TEWL) and changes in skin moisture content were obtained for the experimental duration. At 0, 1 day or 1, 2, 3, 4 weeks after alcohol removal rats were euthanized and their skin was analyzed for alcohol and aldehyde dehydrogenase, and lipid peroxidation. Transdermal penetration of the herbicide paraquat, industrial solvent dimethyl formamide (DMF), insect repellant N,N-diethyl-m-toluamide (DEET) and herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was also determined. Transdermal absorption, LDV, TEWL, skin alcohol and aldehyde dehydrogenase, as well as lipid peroxidation significantly increased after continuous ethanol exposure (p<0.05). These factors remain elevated for up to four weeks after termination of ethanol consumption, showing that skin changes induced by alcohol are not immediately reversible and reflect fundamental changes in the skin itself. This work provides a starting point for examining the link between ethanol ingestion and skin disorders associated with alcohol use.

The effects of chronic exposure to ethanol and cigarette smoke on the formation of peroxynitrite, level of nitric oxide, xanthine oxidase and myeloperoxidase activities in rat kidney

The aim of this study was to investigate the effects of chronic ethanol intake and cigarette smoke exposure on rat kidney. The animals were divided into four experimental groups: (1) the control group (C), (2) the ethanol group (E), (3) the cigarette smoke group (CS), and (4) the cigarette smoke plus ethanol group (CS+E). Rats in E, CS and CS+E groups were treated with ethanol and/or cigarette smoke for 6 months. The animals were killed and the kidneys were removed to determine the activity of xanthine oxidase (XO), myeloperoxidase (MPO) and the levels of nitric oxide (NO). Histopathological and immunohistochemical analysis were performed in kidney tissues. The activity of XO/g protein were 2.8 +/- 0.3, 5.2 +/- 0.3, 3.2 +/- 0.1, and 7.4 +/- 0.7 U for C, E, CS and CS+E groups, respectively. In groups E, and CS+E, the XO values were significantly higher than in group C (P < 0.05). The increase in XO activity of CS was not significantly different from group C (P > 0.05). There was a significant increase in XO activity of group CS+E as compared to CS and E groups (P < 0.05), and also a significant difference in XO activity between E and CS was observed (P < 0.05). The activity of MPO/g protein were 13.5 +/- 0.6, 16.2 +/- 1.1, 14.7 +/- 1.1, 23.8 +/- 0.9 U for C, E, CS, and CS+E groups, respectively. While MPO activity of kidneys from group CS+E were significantly higher as compared to C, CS, and E groups (P < 0.05), there was no significant difference among the groups of C, CS, E (P > 0.05). The levels of NO/g wet tissue were 347.7 +/- 8.5, 261.1 +/- 4.8, 329.8 +/- 5.6, and 254.2 +/- 3.8 nmol for C, E, CS, and CS+E groups, respectively. In groups of E and CS+E, the NO values were significantly lower than that of group C animals (P < 0.05). Although we detected lower NO levels in the E and CS+E groups than in CS group (P < 0.05), a significant difference in NO levels between CS+E and E groups was not observed. In the histopathological analysis of the kidney slices, severe degenerations in kidney tissues of group CS, E, CS+E were observed. Generally, the histological changes in kidney of CS+E and E groups were more severe than those observed in CS alone. While we observed a strong immunoreactivity for anti-nitrotyrosine antibody in kidneys of group CS+E, examination of sections from rat kidneys in group E revealed moderate staining. On the other hand, group CS had very little immunostaining. There was no immunostaining in group C. We concluded that chronic ethanol administration and cigarette smoke exposure may cause oxidative and nitrosative stress which lead to rat kidney damage.

Effects of Chronic Ethanol Ingestion and Folate Deficiency on the Activity of 10-Formyltetrahydrofolate Dehydrogenase in Rat Liver

We recently observed that ethanol feeding impairs 10-formyltetrahydrofolate (10-FTHF) dehydrogenase (EC 1.5.1.6.) and 10-FTHF hydrolase activity in rats. In the present study, we explored the effects of folate deficiency or sufficiency combined with alcoholic intake on 10-FTHF and possible mechanisms by which chronic ethanol ingestion produces folate deficiency. Sprague-Dawley rats were fed either folate-sufficient (FS) or folate-deficient (FD) diets; with or without ethanol (E) for four weeks. Hepatic 10-FTHF dehydrogenase and hydrolase activity, plasma folate and homocysteine were measured at baseline and after feeding experimental diets. Liver weight increased slightly with either folate deficiency or ethanol consumption. In rats fed the folate-sufficient diet with ethanol (FSE), plasma folate was decreased slightly (p<0.05) and plasma homocysteine elevated compared to rats fed the FS diet without ethanol. Ethanol did not affect plasma folate and plasma homocysteine in FD rats. Red-blood cell (RBC) folate was increased similarly in rats by ethanol feeding (FSE and FDE>FS and FD). Feeding folate deficient or ethanol (FSE, FD and FDE) diets depressed hepatic activities of 10-FTHF dehydrogenase, which catalyzes the oxidative deformylation of 10-FTHF to tetrahydrofolate (THF) and carbon dioxide. Rats consuming the FDE diet had the lowest enzyme activities of the experimental groups, implying that folate deficiency and ethanol consumption each affect enzyme activity. We confirm that ethanol decreases hepatic 10-FTHF dehydrogenase activity and show that this decrease occurs irrespective of folate status. This shows that modulation of 10-FTHF is one possible mechanism by which ethanol intake decreases folate status and affects one-carbon metabolism.

Effect of chronic ethanol ingestion on Purkinje and Golgi cell firing in vivo and on motor coordination in mice

As motor coordination impairment is a common symptom of acute and chronic alcohol intoxication, different studies have been conducted on cerebellar Purkinje cell sensitivity to ethanol since Purkinje cell firing constitutes the final integrative output of the cerebellar cortex. However, the effects of chronic ethanol ingestion on Purkinje firing and other cerebellar neurons such as Golgi cells remain unknown. Here, we studied the extracellular discharge of Purkinje and Golgi cells in four groups of non-anesthetized mice drinking ad libitum either 0%, 6%, 12% or 18% ethanol isocallorically compensated with sucrose 25% during a 3-month period. No difference in Golgi cell firing was found with respect to ethanol consumption. The only group that presented significant differences in Purkinje cell firing compared to the other groups was the 18% ethanol-drinking group. These mice presented decreased simple spike and complex spike firing and increased complex spike duration and pause. The 18% ethanol-drinking group was also the only one to present a slight but significant motor coordination impairment (evaluated by rotarod and runway) in naïve task. No motor coordination impairment was noticed in task learned before ethanol consumption. These results suggest that chronic high doses of ethanol are necessary to produce Purkinje cell firing alterations and measurable motor coordination impairment in naïve task. These alterations in Purkinje cell firing did not affect the ability to learn or to recall a motor coordination task.